Many cellular processes are controlled by changes in cell membrane potential due to the action of carrier proteins and ion channels. Carrier proteins bind specific solutes and transfer them across the lipid bilayer of biological cell membranes by undergoing conformational changes that expose the solute binding site sequentially on one side of the membrane and then on the other.
Unlike carrier proteins, ion channel proteins are transmembrane proteins that form pores in biological membranes which allow ions and other molecules to pass from one side to the other. There are various types of ion channels, for instance, “leak channels,” “voltage-gated channels,” “ligand-gated channels,” and channels modulated by interactions with proteins, such as G-proteins.
Ion channel proteins primarily mediate the permeation of a particular ion. For example, sodium (Na+), potassium (K+), chloride (Cl−), and calcium (Ca2+) channels have been identified. Ion channels are largely responsible for creating the cell membrane potential, which is the difference in the electrical charge on the opposite sides of the cell membrane (B. Alberts et al., supra).
The wide variety of carrier proteins and ion channels represents a rich collection of new targets for pharmaceutical agents. Many chemicals, compounds, and ligands are known to affect carrier protein and/or ion channel activity.
Ion channel activity can be measured using the technique of patch-clamp analysis. The general idea of electrically isolating a patch of membrane using a micropipette and studying the channel proteins in that patch under voltage-clamp conditions was outlined by Neher, Sakmann, and Steinback in “The Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes,” Pflueger Arch. 375; 219-278, 1978.
The patch clamp technique represents a major development in biology and medicine. For example, the technique allows measurement of ion flow through single ion channel proteins, and allows the study of single ion channel responses to drugs. Briefly, in a standard patch clamp technique, a thin glass pipette (with a tip typically about 1 μm in diameter) is pressed against the surface of a cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel proteins in a tiny patch of membrane. The activity of these channels can be measured electrically (single channel recording) or, alternatively, the patch of membrane can be ruptured allowing the channel activity of the entire cell membrane to be measured (whole cell recording).
During both single channel recording and whole-cell recording, the activity of individual channel subtypes can be further resolved by imposing a “voltage clamp” across the membrane. Through the use of a feedback loop, the “voltage clamp” imposes a voltage gradient across the membrane, limiting and controlling overall channel activity and allowing resolution of discrete channel subtypes.
The time resolution and voltage control in such experiments are impressive, often in the msec or even μsec-range. However, a major obstacle of the patch clamp technique as a general method in pharmacological screening has been the limited number of compounds that could be tested per day. In addition, the standard techniques are further limited by the slow rate of sample compound change, and the spatial precision required by the patch-clamp pipettes.
A major factor limiting throughput of the patch clamp technique is the perfusion system, which directs the dissolved test compound to cells and patches. In other words, the cells are perfused (i.e., bathed) in a solution, and the test compound is directed into the solution so that its effect on the cell can be measured. In traditional patch clamp setups, cells are placed in large experimental chambers (0.2-2 ml wells), which are continuously perfused with a physiological salt solution. Chemicals are then applied by switching the chamber inlet to a valve connected to a small number of solution bottles containing the chemical(s). However, this technique has several drawbacks. First, the number of different compounds which may be connected at one time is limited by the number of feeding bottles. Second, the volumes required for test samples and supporting fluid require large amounts of costly chemicals. Third, the time required to change the solute composition around cells and patches remains high, and this is a rate-limiting step. Accordingly, there have been several attempts to increase the throughput capacity of patch-clamp recordings.
The development of sophisticated systems for local application of compounds to activate neurotransmitter-regulated channels, like the U-capillary and other systems, has reduced effective application times. However, the volume of bath solution exchanged by such fast application systems is quite large. Such large volume requirements limit the use of these procedures in the medical industry due to the high costs of reagent and the extensive time required to test tens of thousands of chemicals in varying concentrations. These prior art systems are further limited by the inflexibility and low capacity of the feeding systems that fill the U-capillary, which are virtually identical to the systems used in conventional patch clamp experiments.
U.S. application Ser. No. 09/900,627 filed Jul. 6, 2001 by Weaver et. al. (“Weaver”) discloses a system that can measure electrical properties of cells. The Weaver system does not use a pipette tip to attach to cell membranes. Rather, a plurality of pores on a porous surface attach and seal to a plurality of cell membranes. One side of the porous surface is coupled to a ground electrode, and the other side is coupled to a measuring electrode. In one embodiment where the porous surface is a microchip, each cell may be attached to its own ground and measuring electrodes, allowing for cell-specific measurements. When test solutions are applied to one or more sides of the porous surface, a patch clamp recording can be measured for the attached cells. The system can be automated so that multiple porous surfaces are tested simultaneously on a multi-well plate.
U.S. patent application Ser. No. 10/239,046 (Pub. No. U.S. 2003/0139336 A1) filed Mar. 21, 2001 by Norwood et. al. (“Norwood”) provides an automated system for establishing a patch clamp configuration. Norwood's system is limited to attaching a patch pipette to a cell located at the liquid-air interface of a suspended liquid, such as a drop of liquid suspended from the bottom of a capillary tube. Increasing (or decreasing) pressure inside the tube causes the meniscus, the liquid-air interface, to bulge outward (or inward). In the Norwood system, the cell is outside the patch pipette before it is patched. Also, the air pressure system is applied to a second tube that holds and suspends the cellular liquid; air pressure is not applied to the patch pipette itself.
U.S. Pat. Nos. 6,063,260, 6,117,291, and 6,470,226 to Olesen et. al. (collectively, “Olesen”) disclose a high throughput patch clamp system wherein a computerized motor control system causes a patch pipette to patch a cell automatically selected from a cell bath. The pipette tip and cell then remain affixed in a perfusion chamber for patch clamp measurements. An autosampler controls a valve that alternately directs fluid from various sources into the perfusion chamber, including one or more test chemical solutions and washing solutions. Thus, in the Olesen system a number of tubes and pumps are used to pump test chemicals and washing baths into and out of the perfusion chamber.
U.S. Pat. No. 6,048,722 to Farb et. al. (“Farb”) discloses an automatic patch clamp perfusion system that perfuses patched cells with a plurality of test and wash solutions. The test and wash solutions drain from a plurality of reservoirs through a multi-barrel manifold into a recording chamber, which contains a patched cell. A valve controls which solution perfuses the cell at a given time. As in the Olesen system, the Farb system involves a number of tubes and pumps to pump test chemicals and washing baths into and out of the recording chamber.
The disclosures of Weaver, Norwood, Olesen, and Farb are incorporated herein by reference in their entirety.
There remains a need for faster, cheaper methods of high throughput screening. Such high-throughput screens would be invaluable for the search and identification of agents that modulate ion channel activity. In turn, such agents would be useful for the treatment of various diseases.